Abstract Live attenuated RSV vaccine candidates are grown in Vero cells because these cells support RSV replication better than the several others approved by the WHO. However, during growth in Vero cells, the viral attachment glycoprotein, G, is cleaved and no longer supports infection of primary well differentiated human airway epithelial (HAE) cultures. The RSV G (attachment) glycoprotein on the virion surface is essential for efficient infection of its target cell, the ciliated columnar cells in the HAE cultures. HAE cultures are an excellent in vitro model for in vivo RSV infection of the nasal epithelium of a vaccinee. Inefficient infection of these cells would necessitate that vaccine inocula contain 10-fold more virus than if the G protein were not cleaved, greatly increasing the price of vaccine production. Project 2 will identify mutations that prevent cleavage and insert these mutations into the G gene to prevent G protein cleavage during vaccine production in Vero cells. Candidate `repaired G' viruses will be tested for their infectivity and ability to spread in HAE cultures, stability during repeated passage in Vero cells, and immunogenicity in cotton rats. The best `repaired G' mutations will be inserted into double and triple gene mutant viruses containing the best NS1 protein mutants (predicted to enhance immunogenicity) or/and the best L protein mutants (representing a range of attenuating mutations) that will be generated by Projects 1 and 3, respectively. These viruses will all be tested in HAE cultures (Core C) and in cotton rats (Core B) for function and antibody production using novel ELISA and neutralization assays developed by Core C). Finally, the overall immune response to these mutant RSV vaccines will be assessed with a novel gene expression chip assay developed by Core C in collaboration with Project 4. This assay identifies a `safe' immune response, as one that is similar to mild RSV infections in infants rather than severe RSV disease. The best combination mutant viruses will be selected as ideal live attenuated RSV vaccine candidates for future testing in non-human primates and in human trials.